Rapid single-step formation of liposomes by flow assisted stationary phase interdiffusion.

Laboratory preparation of unilamellar liposomes often involves multiple steps carried out over several hours to achieve a monodisperse size distribution. Here, we present a methodology based on a recently introduced lipid self-assembly principle-stationary phase interdiffusion (SPI)-to prepare large unilamellar vesicles (LUVs) of a monodisperse population in a short period of about 10 min. The stationary interface between a lipid-ethanol phase and an aqueous phase is created by a density difference induced convective flow in a horizontal capillary. The average size of the liposomes, as expected from the SPI principle, is modulated only by the temperature and the type of lipids. Lipid concentration, ethanol content, pH of the aqueous phase, and the time duration of the experiment have little influence on the mean diameter of the vesicles. This simple methodology can be easily carried out with a capillary and a micro-needled syringe and provides a rapid production tool for researchers requiring reproducible liposome suspensions. Refined natural lipids, based on soy and egg lecithin mixtures, yield LUVs in the range 100-200 nm, suitable for drug delivery applications.

Spontaneous formation of single component liposomes from a solution.

The diameter of lipid vesicles is generally known to be determined by parameters external to the system, such as fluid shear, electric fields, co-surfactants, etc. We present a mechanism by which a system consisting of a single component lipid can spontaneously assemble from a solution phase to form monodisperse unilamellar vesicles of well-defined diameters dictated only by thermodynamic parameters intrinsic to the system. Here, the lipids self-assemble as vesicles when an aqueous phase diffusively replaces the original solvent in a macroscopically stationary (or quiescent) manner. We demonstrate this using phosphatidyl choline lipid-ethanol-water systems, where the average diameter of the liposomes is shown to be intrinsic, in reasonable agreement with the Helfrich's model of the vesicle free energy. The size depends only on the temperature and the lipid type, eliminating dependence on kinetic effects or external forcing normally observed. The method provides the first pure system to study the self-assembly of vesicle-forming surfactants; and with a natural thermodynamic length scale, it may have an implication for the vesicle size selection under pre-biotic conditions.

Influence of micro-mixing on the size of liposomes self-assembled from miscible liquid phases.

Ethanol injection and variations of it are a class of methods where two miscible phases-one of which contains dissolved lipids-are mixed together leading to the self-assembly of lipid molecules to form liposomes. This method has been suggested, among other applications, for in situ synthesis of liposomes as drug delivery capsules. However, the mechanism that leads to a specific size selection of the liposomes in solution based self-assembly in general, and in flow-focussing microfluidic devices in particular, has so far not been established. Here we report two aspects of this problem. A simple and easily fabricated device for the synthesis of monodisperse unilamellar liposomes in a co-axial flow-focussing microfluidic geometry is presented. We also show that the size of liposomes is dependent on the extent of micro-convective mixing of the two miscible phases. Here, a viscosity stratification induced hydrodynamic instability leads to a gentle micro-mixing which results in larger liposome size than when the streams are mixed turbulently. The results are in sharp contrast to a purely diffusive mixing in macroscopic laminar flow that was believed to occur under these conditions. Further precise quantification of the mixing characteristics should provide the insights to develop a general theory for size selection for the class of ethanol injection methods. This will also lay grounds for obtaining empirical evidence that will enable better control of liposome sizes and for designing drug encapsulation and delivery devices.